Biomedical subjects
Publications and source records attributed to D W Golde.
Human T lymphocytes transformed by human T cell leukemia-lymphoma viruses or activated by lectins were found to produce stimulating factors that promoted both proliferation and maturation of oligodendroglial and astroglial cells in vitro.
We have identified a human T lymphocyte cell line, the Mo line, that constitutively elaborates a lymphokine that activates macrophage receptors for the third complement component (C3) for phagocytosis. The molecule is physically, functionally, and immunologically very similar, if not identical, to a previously characterized murine lymphokine. Both molecules were inactivated by the same physical and chemical treatments, activated macrophage C3 receptors for phagocytosis, and freed macrophage C3 receptors to move within the cell's plasma membrane. In addition, both the human and the murine products were recognized by the same monoclonal antibodies. Treatment with monoclonal antibodies abolished both the ability of lymphokine-containing supernatants to mobilize macrophage C3 receptors and the supernatants' ability to activate C3 receptor-mediated phagocytosis. The dose-response curves for both effects of the antibodies were identical, strongly suggesting that a single molecule was responsible for both effects and that the lymphokine activated marophage C3 receptors for phagocytosis by freeing the anchored receptors and allowing them to diffuse within the cell's plasma membrane. These findings strengthen the hypothesis that, for a receptor to promote phagocytosis, it must be able to diffuse within the macrophage plasma membrane.
Gamma (immune, type II) interferon is an antiviral and immuno-regulatory glycoprotein usually obtained from mitogen- or antigen-induced peripheral blood or splenic mononuclear cells. A human "helper-inducer" T-lymphocyte cell line (Mo) is an inducible source of gamma interferon. Mo gamma interferon has been biochemically characterized and partially purified, and resembles the gamma interferon species derived from normal cell sources.
Neutrophil migration inhibition factor from T lymphocytes (NIF-T) purified by antibody affinity chromatography and gel filtration chromatography was radioiodinated and identified as a 26,000-MW protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). NIF-T was identified by elution of biological activity from gel fractions and selective adsorption of a radioiodinated mediator by HL60 cells differentiated in the presence of dimethyl sulfoxide (DMSO) to develop receptors for NIF-T. A goat neutralizing antibody for NIF-T neutralized and immunoprecipitated migration inhibition activity in the conditioned medium from Mo T-lymphoblast cells and human peripheral blood lymphocytes (PBL) cultured with concanavalin A (Con A), but not from RPMI 1788 B-lymphoblast cells with leukocyte migration inhibitory factor (LIF) activity. These studies distinguish NIF-T both chemically and immunologically from LIF.
The nucleic acid sequence of the 3' region of human T-cell leukemia virus type II (HTLV-II) proviral DNA was determined using a HTLV-II proviral clone that could be recovered as infectious, transforming virus. The sequence data indicate a region of unknown function of approximately equal to 1.6 kilobase pairs in the 3' region, analogous to the X region previously identified in human T-cell leukemia virus type I (HTLV-I). Three overlapping open reading frames are present in the X region of HTLV-II. One of these open reading frames, Xc, is most likely to encode a protein product, because it has greater predicted amino acid sequence homology (78%) with the X-IV region of HTLV-I and a greater percentage of its base differences with X-IV at the third nucleotide position of codons than do the other open reading frames. Sequences of the X-region that include the open reading frames are conserved in two deletion mutants of HTLV-II, which are associated with a subline of Mo cells with a decreased dependence on fetal bovine serum.
The nucleotide sequence of the human T-cell leukemia virus type II (HTLV-II) long terminal repeat (LTR) and its surrounding regions were determined. Our results show the following structural features: (i) the LTR is 763 base pairs (bp) in length and consists of 314 +/- 1 bp of region U3, 248 +/- 1 bp of region R, and 201 bp of region U5; (ii) the terminal nucleotides in the LTR form an inverted repeat of T-G.....C-A; (iii) 6-bp direct repeats of cellular sequences flanking the provirus were present; and (iv) the putative functional signals for initiation or termination of viral RNA synthesis were identified. Comparison of the HTLV-II LTR sequence with that previously published for adult T-cell leukemia virus (ATLV; HTLV-I) shows that the LTRs are distinct. Some small regions are conserved between HTLV-II and ATLV, involving sequences important for transcription and a sequence of 21 nucleotides repeated three times in U3. This 21-bp repeat may be important in regulating viral transcription in lymphoid cells.
HCL is a lymphoproliferative disorder, primarily of B cells. T cell variant HCL is a rare clinical entity, which has a clinical picture similar to that of the common B cell HCL disease. HTLV-II has been isolated from a case of T cell variant HCL. This subtype of HTLV-II-associated disease is indolent in character in comparison to the very aggressive HTLV-I-associated disease. Investigation of the biology and molecular genetics of HTLV is in progress. It is hoped that better understanding of HTLV and the comparative differences between HTLV-I and HTLV-II will provide specific insights into the mechanisms of human leukemogenesis.
To study the influence of a biologic environment on cultured human leukemia cells, KG-1, KG-1a, and HL-60 cells were inoculated subcutaneously into newborn nude mice. The cells developed myelosarcomas at the site of inoculation and in lungs and kidneys. KG-1 and HL-60 myelosarcomas were successfully passaged through adult nude mice, whereas KG-1a tumors proliferated only after transplantation into newborn hosts. The human nature of the cells forming myelosarcomas in mice was assessed by chromosomal analyses and detection of cross-reactivity with an antibody to the human leukemia cell line K562. We undertook electron microscopic and cytochemical examinations of the cells proliferating in vitro and in the mice. The granules of KG-1 cells in vivo did not react for acid phosphatase, as observed in vitro, and the HL-60 cells proliferating in mice lost the perinuclear myeloperoxidase (MPO) demonstrated in cultured cells. Although the influence of an in vivo selection of cell subpopulations cannot be ruled out, the enzymatic changes are compatible with induced cell differentiation. Conclusive evidence of differentiation in vivo was observed in the KG-1a cell subline. The undifferentiated KG-1a blasts developed cytoplasmic granules and synthesized MPO during proliferation in vivo. These observations indicate that human leukemia cells from established cell lines proliferate in nude mice and may acquire new differentiated properties in response to the in vivo environment.
A man from Chile developed an aggressive mature T cell leukemia associated with marked eosinophilia. The neoplastic lymphocytes were of T helper surface phenotype, and they expressed the p24 and p19 antigens of human T cell leukemia virus (HTLV). A cell line (ME) was established from the patient's peripheral blood cells that was initially composed of eosinophils and T and B lymphocytes. The B lymphocytes of the cell line are polyclonal and contain Epstein-Barr virus (EBV) DNA. Many of the T lymphocytes, a few of the B lymphocytes, and none of the eosinophils express HTLV p19 and p24 antigens. By 6 months of culture, the ME line no longer contained eosinophils. A variant line of ME was established; this variant (ME-2) is notable because the cells (greater than 80%) adhere tightly to the bottom of the culture flask; they do not express T lymphocyte markers, but 30% of the cells contain cytoplasmic mu heavy immunoglobulin chains. These pre-B and null lymphocytes contain p19 and p24 antigens (80% of cells), have the HTLV-I genome, and are able to transform normal T lymphocytes in vitro. We isolated a B lymphocyte clone (11A) from ME that expresses cytoplasmic immunoglobulin (70% of cells) and p19 and p24 antigens (75% of cells), contains the EBV and HTLV genomes, and can transform T lymphocytes from normal volunteers. These data show that B lymphocytes can be infected with HTLV, although no disease of HTLV-infected B lymphocytes has been reported. The T lymphocytes from normal adult peripheral blood were easily immortalized (about 70% efficiency) by cocultivation with lethally irradiated ME cells. Twenty-five of 27 of the transformant lines were composed of T lymphocytes with helper antigens, and two of the lines were of T suppressor antigen phenotype. All the cell lines that were tested constitutively produce lymphokines, including colony-stimulating factor (CSF), erythroid-potentiating activity (EPA), macrophage migration-inhibitory factory (MIF), neutrophil-inhibitory factor (NIF), and differentiation-inducing factor (DIF).
Patients with HCL are subject to a variety of medical problems. Many of these complications are caused by the cytopenias and splenomegaly produced by proliferating neoplastic cells. Infection is a common cause of morbidity in HCL, but it is not clear whether there is an inherent defect in the immune system. The incidence of infection is related to neutropenia and is increased by the administration of cytotoxic drugs and corticosteroids; such drugs should be used cautiously in these patients. Opportunistic or unusual pathogens occur frequently in HCL, but recovery from such infections is the rule if the diagnosis is made early. Marrow hypoplasia is not infrequently seen and may present diagnostic difficulties. Such patients may have a lower tumor burden and clinically milder anemia. Hemorrhagic complications are unusual in HCL, though many patients have platelet function abnormalities. Other medical problems occur with increased frequency in HCL, and failure to recognize them leads to increased morbidity in this disease. Autoimmune disease is seen in up to one fourth of patients. It takes the form of self-limited skin and joint disease, or a more progressive, systemic of patients. It takes the form of self-limited skin and joint disease, or a more progressive, systemic vasculitis. Both forms can usually be treated with splenectomy or corticosteroids, but alkylating agents can also be used successfully. Bone disease is usually localized and responds well to radiotherapy. Other problems such as amyloidosis, multiple myeloma, and paraproteinemia are uncommon in HCL.
In vitro culture of haematopoietic cells has provided some surprising insight into humoral regulation of haematopoietic cell growth. Each stage of haematopoiesis is subject to strict regulatory mechanisms involving humoral modulators. These factors called haematopoietins are a family of polypeptide hormones that specifically regulate the proliferation and differentiation of stem cells giving rise to erythrocytes, granulocytes, monocytes, megacaryocytes, and T and B lymphocytes. Mixed colonies consisting of elements of all haematopoietic lineages can be grown from pluripotent progenitors in vitro. Erythropoietin is the primary regulator of the later stages in erythropoiesis, whereas factors with burst-promoting activity or erythroid-potentiating activity stimulate the growth of the more primitive erythroid cells. The in vitro proliferation and differentiation of granulocytic and macrophage cells is dependent on the stimulation by a granulocyte-macrophage colony-stimulating factor. The mode of action of these regulators can well be studied using the homogeneous cell populations of human myeloid and erythroleukemia cell lines. Observations indicate that these factors are likely to function in vivo as in vitro. Knowledge on the biochemistry and physiology of these factors will have substantial impact on the understanding of human diseases involving abnormal haematopoietic cell growth.
A patient with T-cell chronic lymphocytic leukemia presented with severe megaloblastic anemia with normal serum folic acid and cobalamin concentrations. BFU-E could not be cultured from the patient's peripheral blood unless T-lymphocytes were removed by E-rosette formation. Inhibitory activity by the patient's T-cells was restricted to autologous BFU-E. After cyclic chemotherapy the anemia and megaloblastic changes resolved, peripheral blood BFU-E could be cultured from unfractionated peripheral blood and the T-cell inhibitory activity could no longer be demonstrated. The anemia in this patient is probably due to the neoplastic expansion of a suppressor T-lymphocyte population.
Human myeloid stem cell growth in agar was enhanced by the addition of human plasma, red-cell lysate, hemin, and beta-mercaptoethanol to the culture system. Using conditioned media from a human T-cell or leukocyte-conditioned media as a source of colony-stimulating activity, the number of colonies was increased due to enhancement by an average of 54%. Analysis of colony types indicated that over 80% of the increase in colony number at eight days was due to granulocytic colony growth. Light density marrow cells cultured with enhancing agents produced colony levels of approximately 12-27 CFU-C/2 x 10(3) cells.
Migration-inhibitory-factor (MIF) activity was detected in culture supernatants of the human T-lymphoblast cell line Mo after stimulation with phytohemagglutinin and phorbol myristate acetate. MIF activity was not detected in unstimulated cultures reconstituted with phytohemagglutinin and phorbol myristate acetate. Conditioned medium from the cell line Mo was fractionated by Sephadex G-100 gel filtration. MIF-containing Sephadex fractions corresponding to a Mr of 60,000 to 70,000 were further fractionated by isoelectrofocusing, resulting in a sharp peak of activity with a pI of 4.6 to 5.2. This MIF species constitutes a major form secreted by Mo cells; it adheres to Con A-Sepharose, is trypsin-resistant, and is denser than pure protein as determined by CsCl density gradient centrifugation. These are the same physicochemical characteristics previously established for second-day pH5-MIF from peripheral blood mononuclear cells (W.Y. Weiser et al., J. Immunol. 126, 1958, 1981). In contrast, Sephadex fractions corresponding to larger molecules (Mr 70,000-90,000) contain at least two additional MIF species. These larger MIF forms have a pI of 3.0 to 3.5 and of 4.6 to 5.2 and lack affinity to Con A Sepharose. Thus, the Mo T-cell line produces large quantities of at least three different species of human MIF.
A unique human retrovirus (human T-cell leukemia virus type II, HTLV-II), isolated from a patient with a T-cell variant of hairy-cell leukemia, has been shown to be distinct from the more common isolates of human T-cell leukemia virus. This virus was tested for its ability to transform normal human peripheral blood lymphocytes. The HTLV-II-infected T-cell line Mo-T was lethally x-irradiated and cocultivated with normal human peripheral blood lymphocytes. The cocultivation of normal cells with Mo-T cells resulted in the transformation of the normal cells as evidenced by the establishment of permanent cell lines. The transformed cells are infected with HTLV-II as shown by immunologic tests and molecular hybridization. The cells are of mature T-cell phenotype and constitutively produce lymphokines. An Epstein-Barr virus-transformed lymphoblast B-cell line established from peripheral blood cells of the patient Mo, designated Mo-B, also was found to be infected with HTLV-II. All HTLV-II-infected cells, including the Mo-B cells, were capable of transforming normal cells of T-cell phenotype by transmission of virus by cocultivation. These results indicate that HTLV-II infects both B and T cells but transforms normal human peripheral blood lymphocytes of T-cell phenotype.
The studies reported here were designed to determine whether sera from various patients could prevent neutrophils from responding to the lymphokine, neutrophil migration inhibition factor from T lymphocytes (NIF-T). Neutrophils from healthy donors were treated with sera from 84 subjects and assayed for responses to NIF-T. Serum from 7 of 37 patients (19%) with rheumatoid arthritis, systemic lupus erythematosus, and various forms of vasculitis showed blocking activity. In contrast, none of 47 subjects, including healthy individuals and patients with spondylarthropathies, cancer, and active infections had a serum factor that prevented neutrophils from responding to NIF-T (P less than 0.01). Serum blocking activity occurred transiently in association with infection by Staphylococcus aureus in one patient with rheumatoid arthritis. Moreover, autologous neutrophils from this same patient showed impaired responses to NIF-T. Blocking activity could be eluted from protein A-Sepharose in three of three patients studied. In three of seven patients, blocking activity was detected in serum cryoprecipitates, with a recovery of 46 to 78% of the blocking activity and overall enrichment (purification) of 137- to 281-fold. Analysis of cryoprecipitates by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed the predominance of immunoglobulins M and G. In one patient, the serum blocking activity was not cryoprecipitable, and cryoprecipitates from a patient with essential cryoglobulinemia failed to prevent neutrophils from responding to NIF-T. Blocking activity was relatively specific for NIF-T, as there was no effect on F-met-leu-phe-induced chemotaxis of neutrophils. Serum blocking activity in patients with connective tissue disease showed some correlation (r = 0.50; P less than 0.01) with immune complexes detected by polyethylene glycol precipitation but not Clq binding. These studies suggest that the response of neutrophils to NIF-T may be blocked by serum, possibly as a result of immune complexes or autoantibodies found primarily in patients with connective tissue disease.
Acromegaloidism is a syndrome characterized by features of acromegaly without biochemical evidence of excessive GH or somatomedin production. We searched for a growth factor in the serum of patients with this syndrome. Growth-promoting activity was measured by determining the stimulatory effect of whole and fractionated serum on colony formation by human erythroid progenitors in vitro. Sera from five subjects with acromegaloidism gave a mean (+/- SEM) stimulated colony growth of 211 +/- 4.0 colonies, in contrast to normal sera which yielded a mean colony growth of 100 +/- 11.0 (n = 9; P less than 0.001). When serum was chromatographed on a Sephadex G-200 column, the maximal stimulation of colony growth was found in the fractions coinciding with the descending slope of the second protein peak. Based on gel filtration chromatography, the estimated molecular weight was 70,000 daltons. Epidermal growth factor, nerve growth factor, fibroblast growth factor, and platelet-derived growth factor resulted in no substantial stimulation of colony growth under the conditions used. Although the erythroid progenitor cells of a Laron dwarf were unresponsive to 200 ng/ml human GH, they were clearly stimulated by serum from a patient with acromegaloidism. The present study describes the presence of a heretofore unidentified growth factor in the serum of subjects with acromegaloidism. This factor also stimulated the erythroid precursor cells of a Laron dwarf whose cells were unresponsive to GH. The physiological role of this growth factor in normal man as well as its pathogenic role in subjects with acromegaloidism remain to be established.
An updated analysis was done of 71 patients with hairy cell leukemia originally reported in 1978; 36 patients (51%) had died, 28 (39%) were still alive, and 7 (10%) were lost to follow-up. The median actuarial survival time for the entire group from the onset of symptoms was 70 months. Of the 28 known living patients, 8 had been followed for 12 years or more; the longest survivor has remained alive 289 months after the onset of symptoms. Eleven of the original 71 patients required no therapy; 5 remained alive 5 years or more. Causes of death included progressive disease in 4 patients, infections in 20, hemorrhage in 1, unknown causes in 4, and nonhematologic causes in 7.